A novel assay to evaluate promoting effects of proteins on calcium oxalate crystal invasion through extracellular matrix based on plasminogen/plasmin activity

Abstract

One of the important processes in kidney stone development is crystal invasion through extracellular matrix (ECM). Some proteins in renal tissue or urine have been thought to aggravate crystal invasion. However, this pathogenic mechanism has been previously under-investigated due to a lack of crystal invasion assay. In the present study, we have developed a novel assay for the investigations of calcium oxalate monohydrate (COM) crystal invasion. Matrix gel was loaded into an in-house migration chamber made on a glass slide to simulate the ECM environment. COM crystals were coated with the tested protein, which was then bound with plasminogen. The crystal-protein-(plasminogen) complex and urokinase plasminogen activator (uPA) were placed on-top of the matrix gel. If the tested protein had plasminogen-binding capability, the remaining plasminogen would be activated by uPA to plasmin, which caused crystal migration through the matrix gel. We then applied this novel assay to evaluate effects of some abundant kidney/urine proteins (including purified albumin, carbonic anhydrase, lysozyme and Tamm-Horsfall protein) on COM crystal invasion. The data revealed that albumin, which is the known plasminogen-binding protein, dramatically induced plasmin activity and crystal invasion, whereas other proteins had no significant effects as compared to the control. In summary, we have successfully developed a novel assay for the investigations of crystal invasion based on the plasminogen/plasmin system. This assay is applicable to examine proteins that may serve as potential aggravators of crystal invasion and thus will be very useful for further studies on kidney stone development. © 2012 Elsevier B.V. All rights reserved.